The goal of this proposal is to obtain biochemical and molecular characterization of the aspartyl protease, the thiol protease, and endogenous inhibitor(s) that are involved in processing proenkephalin and beta-protachykinin, and to understand the coordinate repletion of this multicomponent processing enzyme system. These studies are important because it is only the mature enkephalin and substance P peptides, and not their precursors, that function as active neurotransmitters. The processing enzymes that convert the precursors into the active peptides are crucial for the control of neuropeptide biosynthesis. Using a novel approach for production of authentic enkephalin and tachykinin precursor substrates from their cDNA's by in vitro transcription and translation (with 35S-Met), we have identified aspartyl and thiol proteases that show precursor preference. In this proposal, functional sequences of the aspartyl protease will be determined by amino acid sequencing of antibody (monoclonal) epitopes that are important for enzymatic activity, and enzyme structure will be deduced from its cDNA sequence. Biochemical characterization of purified thiol protease (pH, inhibitors, cofactors, and substrate preference) will provide knowledge of its regulation, and immunochemical characterization with monoclonal antibodies can indicate structural homology or heterogeneity with other proteases. It will be important to determine the enzymes' sites of cleavage by amino acid sequencing of proteolytic products (precursors produced by bacterial expression of cDNA's), and to study cleavage site specificity using mutant precursors. Regulation by endogenous inhibitors, including alpha l-antichymotrypsin-related proteins (al -AC), will be studied by characterizing the interactions of purified inhibitors with aspartyl or thiol proteases. The coordinate regulation of this multicomponent processing system will be studied in chromaffin cells during increased enkephalin production. These studies will provide significant advances in understanding the role of processing enzymes and their inhibitors in regulating enkephalin and tachykinin peptide biosynthesis. Determination of al-AC-related protein(s) as a possible inhibitor may implicate a role for the thiol protease in Alzheimer's disease that contains al-AC in amyloid deposits. Knowledge obtained from this project will clearly be important for elucidating molecular mechanisms underlying neurologic diseases, and will provide new insight into therapeutic strategies.